Personalized medicine requires the development of biomarker diagnostic assays, reflecting individual variations and thus allowing tailored therapeutic interventions. Plasma membrane proteins play a key role in various physiological functions and pathological conditions, while currently no proper and simple assays are available for their quantitative determination. Since these proteins undergo complex processing and trafficking, mRNA levels do not correspond to their final expression level in the target membrane. Moreover, neither mRNA nor the final protein levels can be properly quantitated in human tissue samples, due to difficulties in obtaining and processing the most relevant human tissues. There are numerous data for genetic polymorphisms and mutations, potentially affecting membrane protein expression, but data are scarce for actual protein expression levels in the human body. As mentioned above, mRNA expression levels do not coincide with protein expression for most membrane proteins, and human tissue samples are also difficult to obtain. Transporters and receptors responsible for ADME-Tox properties, drug sensitivity, receptor-stimulation and inhibition, or cellular response modulation, corresponding to various diseases and drug treatments, show great individual variations. The exact determination of these variations in particular at the protein level, allowing the advance of personalized medicine, is still lacking.
It has been demonstrated that human erythrocytes (red blood cells, RBC) express numerous membrane proteins, including transporters and receptors in their single plasma membrane [Goodman S. R. et al., “The Human Red Blood Cell Proteome and Interactome” Experimental Biology and Medicine (2007), 232:1391-1408; Pasini, E. et al. (2010). “Red blood cell (RBC) membrane proteomics—Part I: Proteomics and RBC physiology.” J Proteomics 73(3): 403-20]. According to currently available information on the erythrocyte membrane proteome, numerous membrane proteins with known involvement in human diseases and thought to be characteristic for special organs and tissues, are expressed in measurable quantities in the erythrocyte membrane [see Hernández-Hernández A et al., “Alterations in erythrocyte membrane protein composition in advanced non-small cell lung cancer” Blood Cells Mol Dis. (2006) 36(3):355-63; Goodman S. R. et al., see above].
Various techniques have already been applied to measure the function and/or the expression of red cell membrane proteins related to disease conditions. Erythrocyte Na—Li and Na—H countertransport activity was found to predict susceptibility to diabetes and hypertension (see Koren W, et al., Enhanced erythrocyte Na+/H+ exchange predicts diabetic nephropathy in patients with IDDM. Diabetologia. 1998 February; 41(2):201-5; Weder A. B. et al. Erythrocyte Sodium-Lithium Countertransport and Blood Pressure, Hypertension 2003, 41:842-846, Deak B, et al., Diabetes and erythrocyte Na—Li exchanger, Acta Diabetol (2003) 40:9-13).
For example, Sprague R. S. et al. [“Reduced Expression of Gi in Erythrocytes of Humans With Type 2 Diabetes Is Associated With Impairment of Both cAMP Generation and ATP Release” Diabetes (2006) 55 3588-3593] assessing protein expression levels by Western analysis, reported that the expression of the heterotrimeric G-protein Gi is selectively decreased in erythrocytes of type 2 diabetes patients. Antonelou M. H. et al. [“Apolipoprotein J/Clusterin Is a Novel Structural Component of Human Erythrocytes and a Biomarker of Cellular Stress and Senescence” PLoS ONE, (2011) 6(10) e26032 1-9] identified Secretory Apolipoprotein J/Clusterin (sCLU), a chaperone that has been implicated in several pathological conditions, as a component attached to human RBCs. The authors studied the erythrocytic membrane-bound sCLU by using a combination of molecular, biochemical and high resolution microscopical methods. They have concluded that reduced sCLU protein levels are sensitive biomarkers of senescence and cellular stress. Moreover, sCLI is not an integral membrane protein
All these studies used technologies (e.g. Western blot, transport activity measurements, special microscopy techniqes) are only available in specialized research laboratories, and by using these technologies it is inherently very difficult to quantify membrane protein expression. In some cases flow cytometry was also used to detect erythrocyte membrane proteins [see Saison, C. et al, (2012). “Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior.” Nat Genet 44(2): 174-7], but there was no attempt to offer a technology to quantitate this membrane protein expression.
Therefore there is still a need in the art for effective and simple biomarker diagnostic assays based on erythrocyte membrane protein expression, as well as for such assays reflecting individual variations of patients. There is also a need for such assays to allow tailored therapeutic interventions. It further appears that there is a need in the art for an effective and fast method for quantitative assessment membrane protein expression levels in erythrocytes and thereby obtain information on the condition of a subject. The recognition of membrane protein in their native or mildly fixed, membrane embedded form is a great advantage in such a technology. Moreover, it seems that there is no clear proposal in the art for the diagnostic use of membrane protein expression, including membrane receptor, and in particular membrane transporter or ABC transporter expression in the erythrocyte membranes as a reporter of a patient's condition.
The present inventors have recognized that a rapid, reliable and quantitative immunological type assay useful for diagnostic purposes can be performed if red blood cells are maintained as whole cells and thereby the membrane proteins are maintained in their original environment. Thereby membrane protein expression levels can be quantitated by measuring the levels of said proteins in the red blood cell membranes. By using the rapid, sensitive and quantitative membrane protein detection method it was possible to find a correlation of erythrocyte membrane protein expression with genetic mutations, polymorphisms, as well as drug-, and cellular responses. It has been found that the potential reflection of tissue-specific membrane protein expression in the erythrocyte membrane make this platform feasible for simple and rapid quantitative biomarker reporter assays. It has also been found that the relatively slow turnover and long life-span of the human red cells (about 120 days) makes this system a stable, relatively slowly responding biomarker platform, best reflecting individual variations or chronic alterations in membrane proteins.